CN104119430A - Plant drought resistance related protein, coding gene and application thereof - Google Patents
Plant drought resistance related protein, coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a plant drought resistance related protein, a coding gene and application thereof. The protein is called BhSMP1, is derived from Boea hygrometrica of gesneriaceae, is as follows (a) or (b): (a) is a protein comprising an amino acid sequence shown as sequence 2 of a sequence table; (b) is a sequence 2 derived and plant drought resistance related protein which is obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown as the sequence 2 of the sequence table. Experiments prove that the invention screens a drought induced expressed BhSMP1 gene, and a protein encoded by the gene, the protein and the coding gene have important theoretical and practical significance for the cultivation of crops, forest grass and other new varieties with improved drought resistance and salt resistance, and can be used for the cultivation and identification of resistant plant varieties desired by agriculture and animal husbandry and ecological environment governance.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of albumen relevant to drought tolerance in plants and encoding gene and application.
Background technology
Along with the shortage of global water resources and the growth of extreme weather events, the yield and quality of arid on crop affect increasingly significant.Therefore, aobvious important by the drought resistance day of molecule manipulation technology raising crop.
Organism is subject to, after drought stress or other poor environment factor, can resisting bad coercing by different approaches.In seed maturity process, be also accompanied by the acquisition of this drought tolerance, comprise synthetic and accumulation, the generation of Protective substances, the removing of objectionable impurities etc. of drought-resistant associated protein.The formation of the content of seed maturity albumen and the drought tolerance of plant has vital role, and can improve the germination rate in adverse circumstance.
Resurrection plant is because of its condition that can stand Extreme drought, can recover very soon the characteristic of normal activities again and be considered to the contrary mechanism of Effect of Anti and the fabulous plant resources of adversity gene is provided in the time that moisture content is sufficient.Revolving capsule lettuce tongue (Boea hygrometrica) is a kind of Gesneriaceae resurrection plant distributing in China, the blade of this plant has very strong drought-enduring recovery ability, under the condition that is 0 at room temperature, relative air humidity, grow after 72 hours, blade relative water content is reduced to 3% left and right, blade area shrinkage is to below 1/3 of former blade area, and photosynthesis stops substantially.If feedwater again, the blade stretching, extension that just can absorb water, and revert to the untreated front apparent state of blade and physiological status (comprising photosynthetic recovery).
Summary of the invention
An object of the present invention is to provide a kind of albumen relevant to drought tolerance in plants and encoding gene thereof.
Albumen provided by the invention, is called BhSMP1, and what derive from Gesneriaceae revolves capsule lettuce tongue (Boea hygrometrica), is following (a) or (b):
(a) protein being formed by the aminoacid sequence shown in sequence in sequence table 2;
(b) replacement and/or disappearance and/or interpolation and the protein that by sequence 2 derived relevant to plant stress tolerance through one or several amino-acid residue by the aminoacid sequence shown in sequence in sequence table 2.
In above-mentioned albumen, the replacement of described one or several amino-acid residue and/or disappearance and/or interpolation refer to replacement and/or disappearance and/or the interpolation of no more than ten amino-acid residues.
The DNA molecular of above-mentioned albumen of encoding is also the scope of protection of the invention.
Above-mentioned DNA molecular is any one DNA molecular in following (1)-(3):
(1) coding region be in sequence table sequence 1 from the DNA molecular shown in 5 ' end 36-272 position Nucleotide;
(2) the DNA sequence dna hybridization limiting with (1) under stringent condition and the DNA molecular of coding and plant stress tolerance correlative protein;
(3) DNA sequence dna limiting with (1) at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have a DNA molecular of 99% homology and coding and plant stress tolerance correlative protein.
Above-mentioned DNA molecular also can be prepared as follows: with primer 5 '-AGGATCCTTAAGGATGTCGGGAGCT-3 ' and primer 5 '-TGAATTCCTTAAGCTGTGCCCGTG-3 '; Taking the DNA molecular shown in the sequence 1 in sequence table as template, the PCR product that amplification obtains, then pass through BamH I and EcoR I double digestion, obtain DNA molecular.
Above-mentioned stringent condition is at 6 × SSC, in the solution of 0.5%SDS, under 65oC, hybridizes, and then uses 2 × SSC, 0.1%SDS and 1 × SSC, and 0.1%SDS respectively washes film once.
Sequence 1 in above-mentioned sequence table is by 443 based compositions, and its open reading frame (ORF) is from 5 ' end 36-272 bit base, and coding has the BhSMP1 of the aminoacid sequence of sequence 2 in sequence table.
Recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contains above-mentioned DNA molecular is also the scope of protection of the invention.
Above-mentioned recombinant vectors is that above-mentioned DNA molecular is replaced to the LEA fragment in expression vector Bin19-35S-LEA-polyA, obtains expressing the recombinant vectors of above-mentioned albumen.In an embodiment of the present invention, above-mentioned recombinant vectors specifically can be BamH I and the EcoR I enzyme of above-mentioned DNA molecular replacement Bin19-35S-LEA-polyA is cut to the recombinant plasmid that the small segment (LEA fragment) between recognition site obtains.Above-mentioned DNA molecular is for being prepared as follows: with primer 5 '-AGGATCCTTAAGGATGTCGGGAGCT-3 ' and primer 5 '-TGAATTCCTTAAGCTGTGCCCGTG-3 '; Taking the DNA molecular shown in the sequence 1 in sequence table as template, the PCR product that amplification obtains, then pass through BamH I and EcoR I double digestion, obtain DNA molecular.
The primer pair of above-mentioned BhSMP1 full length gene or its arbitrary fragment of increasing also belongs to protection scope of the present invention.
Can be any one double base agrobacterium vector or can be used for the carrier etc. of plant micropellet bombardment for the carrier that sets out that builds described plant expression vector, as pBin19, pBI121, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300 or other derivative plant expression vector.
While using the gene constructed recombinant expression vector of BhSMP1, can before its transcription initiation Nucleotide, add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general raw plain gene Ubiquitin promotor (pUbi) etc., they can be used alone or are combined with other plant promoter; In addition, while using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.
For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, can in plant, express antibiotic marker thing (gentamicin marker, kantlex marker etc.) or the anti-chemical reagent marker gene (as anti-weedkiller gene) etc. that can produce the enzyme of colour-change or the gene of luminophor (gus gene, GFP gene, luciferase genes etc.), there is resistance as added.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Above-mentioned albumen, above-mentioned DNA molecular or the application in regulating plant resistance of reverse of above-mentioned recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention; Be specially raising plant stress tolerance in regulating plant resistance of reverse described in above-mentioned application; Described resistance of reverse is specially drought tolerance; Above-mentioned drought tolerance embodies by following: under PEG2000 coercion, the germination rate of transgenic plant seed is greater than described object plant.Above-mentioned plant is specially dicotyledons or monocotyledons, and above-mentioned dicotyledons is further specially Arabidopis thaliana.
Another object of the present invention is to provide a kind of method of cultivating transgenic plant, for the DNA molecular of the above-mentioned albumen of coding is imported to object plant, obtains transgenic plant, and the resistance of reverse of described transgenic plant is higher than described object plant.
In aforesaid method, described resistance of reverse is drought tolerance;
The DNA molecular of above-mentioned albumen of encoding imports object plant by above-mentioned recombinant vectors.
In aforesaid method, described object plant is dicotyledons or monocotyledons, and described dicotyledons is specially Arabidopis thaliana.
Above-mentioned drought tolerance embodies by following: under PEG2000 coercion, the germination rate of transgenic plant seed is greater than described object plant.
Carry and the conventional biological method such as of the present inventionly can lead by Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity, agriculture bacillus mediated to the plant expression vector of plant drought, the relevant protein coding gene BhSMP1 of anti-salt and be transformed in vegetable cell or tissue.The plant host being converted can be both the farm crop such as paddy rice, wheat, soybean, tobacco, corn, rape, Chinese sorghum, cotton, can be also the fruits and vegetables flower plants such as the herbages such as clover, trifolium, wheatgrass and strawberry, tomato.
Of the present invention experimental results show that, the present invention is revolved and capsule lettuce tongue (Boea hygrometrica), is screened a BhSMP1 gene that is subject to drought-induced expression from resurrection plant, this gene is imported to Arabidopis thaliana, obtain turning BhSMP1 Arabidopis thaliana, turn the drought tolerance of BhSMP1 Arabidopis thaliana apparently higher than not genetically modified Arabidopis thaliana, illustrate that BhSMP1 is the albumen relevant to drought resistance in plants.Therefore, this albumen and encoding gene thereof have important theory and practical significance for the new variety such as crop, woods grass of cultivating drought tolerance raising, can be used for cultivation and the qualification of the required resistance plant kind of husbandry and ecological environment treatment.
Below in conjunction with specific embodiment, the present invention will be further described.
Brief description of the drawings
Fig. 1 is that BhSMP1 gene is at the expression revolving in the arid recovery process of capsule lettuce tongue (Boea hygrometrica)
Fig. 2 is the RT-PCR detected result that T2 generation turns BhSMP1 Arabidopis thaliana
Fig. 3 turns PEG8000 tolerance qualification in BhSMP1 Seed Germination of Arabidopsis Pumila process T2 generation
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
The acquisition of embodiment 1, BhSMP1 gene and the expression in drought stress is processed
One, the acquisition of BhSMP1 gene
Extract and (earth culture seedling is carefully cleaned up through drought stress, be put in seasoning in illumination box, temperature: 22.0 DEG C, humidity: 50%RH, photoperiod: 16 illumination/8 dark) process 8 hours revolve capsule lettuce tongue (Boea hygrometrica, be documented in as in Publication about Document: Deng Xin, Hu Z, Wang H (1999): the mRNA differential display visualized by silver staining tested on gene expression in resurrection plant Boea hygrometrica.Plant Mol Biol Rep17:279. public can obtain from Institute of Botany, Chinese Academy of Sciences) total RNA of blade, utilize
library construction test kit (Stratagene, La Jolla, CA) construction cDNA library.Therefrom 4800 genes of random choose, use BioGrid robot (BioRobotics Ltd, Cambridge, UK) automatization point sample at Hybond-N
+nylon membrane (Amersham Biosciences, Freiburg, Germany) is upper, makes cDNA microarray (cDNA chip).By this cDNA chip respectively with process without arid normal growth revolve capsule lettuce tongue blade and process the polyA-RNA that revolves capsule lettuce tongue blade of 8 hours through drought stress prepared
33the probe of P mark is hybridized, and analyzes their dynamic changes in normal condition and drought-induced rear genetic expression.Found that there is 1 gene through drought-induced rise.
Extract through drought stress and process the total RNA that revolves capsule lettuce tongue (Boea hygrometrica) blade of 8 hours and taking it as template, use gene-specific primer GSP-Race1:5 '-GGCGTCCTCCACCTTTTCTT-3 ' to carry out reverse transcription.Reaction system is: total l) 1.0 μ l of RNA(2 μ g/ μ, GSP-Race1(1 μ M) 2 μ l, dNTP(2mM) 5 μ l, DEPC-H
2o6.0 μ l, 5 × M-MLV buffer4 μ l, RNase inhibitor (5u/ μ l, purchased from Takara company) 1 μ l, M-MLV(is purchased from Promega company) 1 μ l.65 DEG C of 10min again after 37 DEG C of reaction 1h, purifying adds C tail after reclaiming (three rich PCR products reclaim test kit).Reaction system is: cDNA5 μ l, DEPC-H
2o13.5 μ l, 5 × buffer5 μ l, dCTP(10mM) 0.5 μ l.75 DEG C of reactions add 1 μ l TdT(purchased from takara company after 5min), then 37 DEG C of reaction 1h, obtain adding the cDNA sequence of C tail.
Taking the cDNA sequence that adds C tail of above-mentioned acquisition as template, carry out pcr amplification.Reaction system used is: cDNA1 μ l, 10 × PCR buffer1 μ l, dNTP(2mM) 1 μ l, primer GSP-Race2:5 '-GAATCGGTCCAGGTCATCTTTAC-3 ' (10 μ M) 0.5 μ l, poly G anchor primer 5 '-GGCCACGCGTCGACTAGTACG-3 ' (10 μ M) 0.5 μ l, Taq enzyme (purchased from takara company) 0.1 μ l.Reaction conditions is: first 95 DEG C of denaturation 4min, and then 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, then 72 extend 2 minutes, totally 35 circulations; Last 72 DEG C are extended 10 minutes.PCR product is carried out to agarose gel electrophoresis detection, and result obtains the band of 450bp left and right; Reclaim the band of this 450bp left and right, with carrier pGEM-T(purchased from promega company) check order after being connected, obtain the PCR product of 443bp.
According to sequencing result design primer 5 '-AGGATCCTTAAGGATGTCGGGAGCT-3 ' and primer 5 '-TGAATTCCTTAAGCTGTGCCCGTG-3 '; Process cDNA that total RNA reverse transcription of revolving capsule lettuce tongue (Boea hygrometrica) blade of 8 hours obtains as template (also can taking the DNA molecular shown in the sequence 1 in sequence table as template) taking drought stress, carry out pcr amplification with above-mentioned primer, reaction system is: cDNA1 μ l, 10 × PCR buffer1 μ l, dNTP(2mM) 1 μ l, the each 0.5 μ l of primer, Taq enzyme (purchased from takara company) 0.1 μ l.Program is: 95 DEG C of denaturation 4min, and 94 DEG C of sex change 45 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, totally 27 circulations; Finally again 72 DEG C extend 10 minutes.
Obtain the product of 250bp left and right, through order-checking, this PCR product has in sequence table sequence 1 from the Nucleotide of 5 ' end 36-272 position, this PCR product is BhSMP1 gene complete sequence, analyze the open reading frame (ORF), 5 '-UTR (5 '-non-translational region) and the 3 '-UTR that find that BhSMP1 gene complete sequence comprises complete BhSMP1 gene, its BhSMP1 gene ORF is sequence 1 deoxyribonucleotide from 5 ' end 36-272 position, proteins encoded called after BhSMP1, the amino acid residue sequence of this albumen is as shown in sequence in sequence table 2.
Two, BhSMP1 gene is at the expression revolving in the arid recovery process of capsule lettuce tongue (Boea hygrometrica)
Carry out 8 kinds of arid processing in various degree to revolving capsule lettuce tongue, be respectively: Fresh Plants (F), in Fresh Plants soil at a slow speed arid 5 days (not watering water treatment 5 days, SD5d), in Fresh Plants soil at a slow speed arid 14 days (not watering 14 days SD14d of water treatment).Extract respectively total RNA, reverse transcription acquisition cDNA that capsule lettuce tongue blade is revolved in 3 kinds of processing.Use gene-specific primer BhSMP1-f5 '-AGGATCCTTAAGGATGTCGGGAGCT-3 ', and BhSMP1-r5 '-TGAATTCCTTAAGCTGTGCCCGTG-3 ' carries out Q-PCR amplification.And taking 18S rRNA gene as internal reference, amplimer is 18S-f5 '-CTTAGTTGGTGGAGCGATTTG-3 ', and 5 '-CCTGTTATTGCCTCAAACTTCC-3 '.Reaction system is: cDNA1 μ l, 10 μ l2 × SYBR Green Master Mix, the each 0.5 μ l of primer, dd water 8.5 μ l.Program is: 95 DEG C of denaturations 30 seconds, and 95 DEG C of sex change 5 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 minutes, totally 50 circulations.
As shown in Figure 1, wherein, F represents the fresh untreated capsule lettuce tongue of revolving to result, and SD5d, SD14d represent respectively to dewater 5 days and 14 days, can find out that this gene is obviously subject to drought-induced.
Embodiment 2, the acquisition that turns BhSMP1 Arabidopis thaliana and functional study thereof
One, turn the acquisition of BhSMP1 Arabidopis thaliana
1, the acquisition of recombinant vectors pBin19-BhSMP1
With primer 5 '-AGGATCCTTAAGGATGTCGGGAGCT-3 ' and primer 5 '-TGAATTCCTTAAGCTGTGCCCGTG-3 '; Taking the DNA molecular shown in the sequence 1 in sequence table as template, obtain the PCR product of 258bp.
The concrete construction process of recombinant vectors pBin19-BhSMP1 is as follows:
1) by the PCR product of 258bp through BamH I and EcoR I(purchased from the precious biotech firm in Dalian) after double digestion, reclaim the fragment (have in sequence table sequence 1 from the Nucleotide of 5 ' end 36-272 position) of about 258bp;
2) by the carrier B in19-35S-LEA-polyA(Liu X. containing CaMV35Sq promotor and pA, Wang Z, Wang L.L., Wu R.H.Phillips J.and Deng X.LEA4group genes from the resurrection plant Boea hygrometrica confer dehydration tolerance in transgenic tobacco, Plant Science176 (2009) 90 – 98; The public can obtain from Institute of Botany, Chinese Academy of Sciences) cut LEA fragment with BamH I and EcoR I enzyme, reclaim the carrier framework of about 13kb, by carrier framework and 1) fragment of the 258bp that obtains is connected, obtain recombinant vectors pBin19-BhSMP1(and cut checking with BamH I and EcoR I enzyme, obtain 258bp positive).
Through order-checking, this recombinant vectors pBin19-BhSMP1 is by the small segment (LEA fragment) between BamH I and the EcoR I restriction enzyme site of the fragment of 258bp (have in sequence table sequence 1 from the Nucleotide of 5 ' end 36-272 position) replacement vector Bin19-35S-LEA-polyA, the carrier obtaining.
This recombinant vectors pBin19-BhSMP1 is subject to 35S promoter (Odell, JT; Nagy, F; Chua, NH.Identification of DNA sequences required for activity of the cauliflower mosaic virus35S promoter.Nature.313 (2005): 810 – 812) control.
2, the acquisition of recombinant bacterium
The above-mentioned 1 recombinant vectors pBin19-BhSMP1 obtaining is transformed into Agrobacterium Gv3101 cell, and (public can obtain from Institute of Botany, Chinese Academy of Sciences, be documented in as in Publication about Document: Holsters M, Silva B, Van Vliet F, Genetello C, De Block M, Dhaese P, Depicker A, Inz6D, Engler G, Villaroel R, Van Montagu M, Schell J (1980) The functional organization of the nopaline A.tumefaciens plasmid pTiC58.Plasmid3:212-230) in, with containing 50 μ g/ml sulphuric acid kanamycins, the YEB solid plate substratum of 50 μ g/ml gentamicins and 50 μ g/ml Rifampins screens, obtain transformant, extract the plasmid of transformant and send to order-checking, plasmid is pBin19-BhSMP1, by the transformant called after recombinant bacterium Gv3101/pBin19-BhSMP1 that contains this plasmid.
3, turn acquisition and the Molecular Identification of BhSMP1 Arabidopis thaliana
1) turn the acquisition of BhSMP1 Arabidopis thaliana
Adopt floral organ infusion method to transform wild-type Arabidopis thaliana Col-0(ecotype columbia, Arabidopsis thaliana recombinant bacterium Gv3101/pBin19-BhSMP1; The public can obtain from Institute of Botany, Chinese Academy of Sciences, is documented in as in Publication about Document: Arabidopsis, a useful weed.Meyerowitz EM, Cell(1989) 56:263-270.) flower, obtain T0 generation and turn BhSMP1 Arabidopis thaliana.Method for transformation is as follows: recombinant bacterium 50uL is inoculated in the YEB substratum of 50mL containing 35mg/L Rifampin, 50mg/L gentamicin sulphate, 50mg/L kantlex, 28 DEG C, 160rpm shakes to OD value 1.5 left and right, centrifugal collection thalline, resuspended with 5% sucrose of same volume, and add 10uL penetration-assisting agent, thaliana flower is dipped in heavy spinning liquid and is soaked 1 minute.Complete to soak and spend rear black plastic bag shading 1 day of using, within second day, recover natural lighting, obtain T0 for turning BhSMP1 Arabidopis thaliana.
In T0 generation, is turned to the selfing of BhSMP1 Arabidopis thaliana, collect T1 for turning BhSMP1 Arabidopis thaliana seed, getting 100 is seeded on the MS substratum that contains 100 μ g/ml sulphuric acid kanamycins, resistance on the above-mentioned MS substratum that contains 100 μ g/ml sulphuric acid kanamycins is separated than the T1 for 3:1 and moves to hot-house culture for the surviving seedling that turns BhSMP1 Arabidopis thaliana, collect seed, sowing, obtains 3 T2 altogether for turning BhSMP1 Arabidopis thaliana homozygous lines: OE10-4, OE26-3, OE31-2.
2) turn the Molecular Identification of BhSMP1 Arabidopis thaliana
In above-mentioned 3 T2 generation of extracting 50 days seedling ages, turns BhSMP1 Arabidopis thaliana homozygous lines: total RNA of the fruit pod of OE10-4, OE26-3, OE31-2, reverse transcription is that cDNA is template, with SMP1-f2
5 ' AGGATCCTTAAGGATGTCGGGAGCT-3 ' and SMP1-r2
5 ' TGAATTCCTTAAGCTGTGCCCGTG-3 ' carry out RT-PCR for primer, in contrast with not genetically modified wild-type Arabidopis thaliana (WT) simultaneously.
Reference gene is Actin, and primer is actinF:5 '-AGGAACACCCTGTTCTCCTGACTG-3 ', actinR:5 '-CAGAGAAAGCACAGCCTGGATAG-3 '.
Pcr amplification program is: 95 DEG C of denaturation 4min, and 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, totally 28 circulations; Finally again 72 DEG C extend 10 minutes; Simultaneously with actin gene as internal reference, pcr amplification program is: 95 DEG C of denaturation 4min, 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, totally 28 circulations; Finally again 72 DEG C extend 10 minutes.
PCR product is carried out to agarose gel electrophoresis detection, every group of sample carries out 2 times to be repeated, result as shown in Figure 2, can find out, in not genetically modified wild-type Arabidopis thaliana (WT), do not amplify band, in T2 generation, turns BhSMP1 Arabidopis thaliana homozygous lines: in OE10-4, OE26-3, OE31-2, all have 258bp goal gene BhSMP1 to express.Further prove, in T2 generation, turns the positive transfer-gen plant of BhSMP1 Arabidopis thaliana homozygous lines.
Adopt and use the same method, empty carrier Bin19-35S-LEA-polyA is proceeded in wild-type Arabidopis thaliana, obtain T0 for turning pBin19 Arabidopis thaliana; In T0 generation, is turned to pBin19 Arabidopis thaliana sowing, sowing, turn pBin19 Arabidopis thaliana until obtain T2 generation.
Two, turn the drought-enduring research of BhSMP1 Arabidopis thaliana
Thereby detecting T2 generation turns in BhSMP1 Seed Germination of Arabidopsis Pumila process and PEG8000 tolerance to be embodied to it is drought-enduring:
Will results 3 strain T2 generations obtaining above-mentioned one after January turn BhSMP1 Arabidopis thaliana homozygous lines: the seed that OE10-4, OE26-3, OE31-2, T2 generation turn pBin19 Arabidopis thaliana and wild-type Arabidopis thaliana broadcasts sowing respectively uniformly in containing 15%(quality percentage composition), 20%(quality percentage composition) in the sprouting box of PEG8000,4 DEG C of vernalization are after 4 days, cultivate under the condition of 22 DEG C, 50% humidity, illumination 16h and dark 8h, add up germination rate every day from cultivating beginning first day, after 5 days, take a picture and complete statistics.Experiment is established three repetitions, 200 seeds of each strain at every turn repeating altogether.
As shown in Figure 3, A is the germination rate statistical graph containing Arabidopis thaliana in the sprouting box of 15%PEG to result, and B is the germination rate statistics containing Arabidopis thaliana in the sprouting box of 20%PEG, and C is the sprouting photo containing Arabidopis thaliana in the sprouting box of 15%PEG;
In T2 generation, turns BhSMP1 Arabidopis thaliana homozygous lines OE10-4 and is respectively 86.91%, 95.92%, 97.83%, 99.85%, 99.85% at the germination rate of cultivating the 1st, 2,3,4,5 days;
In T2 generation, turns BhSMP1 Arabidopis thaliana homozygous lines OE26-3 and is respectively 75.63%, 89.31%, 90.68%, 94.12%, 94.72% at the germination rate of cultivating the 1st, 2,3,4,5 days;
In T2 generation, turns BhSMP1 Arabidopis thaliana homozygous lines OE31-2 and is respectively 72.13%, 86.77%, 89.77%, 97.83%, 99.03% at the germination rate of cultivating the 1st, 2,3,4,5 days;
Wild-type Arabidopis thaliana (col) is respectively 30.23%, 46.85%, 55.53%, 61.13%, 62.63% at the germination rate of cultivating the 1st, 2,3,4,5 days.
T2 is for turning pBin19 Arabidopis thaliana and wild-type Arabidopis thaliana without significant difference.
As can be seen from Figure 3, T2 generation turns BhSMP1 Arabidopis thaliana seed to be compared than wild-type Arabidopis thaliana, under the coercing of PEG8000, germination rate improves, illustrate that T2 has very strong resistance to PEG for turning BhSMP1 Arabidopis thaliana, show, in T2 generation, turns BhSMP1 Arabidopis thaliana and has very strong drought tolerance.
Result shows, the drought tolerance that T2 generation turns BhSMP1 Arabidopis thaliana is obviously better than wild-type Arabidopis thaliana, illustrates that BhSMP1 is the albumen relevant to drought tolerance in plants.
Claims (10)
1. an albumen is following (a) or (b):
(a) protein being formed by the aminoacid sequence shown in sequence in sequence table 2;
(b) replacement and/or disappearance and/or interpolation and the protein that by sequence 2 derived relevant to plant stress tolerance through one or several amino-acid residue by the aminoacid sequence shown in sequence in sequence table 2.
2. the DNA molecular of albumen described in coding claim 1.
3. DNA molecular as claimed in claim 2, is characterized in that: described DNA molecular is any DNA molecular in following (1)-(3):
(1) coding region be in sequence table sequence 1 from the DNA molecular shown in 5 ' end 36-272 position Nucleotide;
(2) the DNA sequence dna hybridization limiting with (1) under stringent condition and the DNA molecular of coding and plant stress tolerance correlative protein;
(3) DNA sequence dna limiting with (1) at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have a DNA molecular of 99% homology and coding and plant stress tolerance correlative protein.
4. contain recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium of DNA molecular described in claim 2 or 3.
5. recombinant vectors as claimed in claim 4, is characterized in that:
Described recombinant vectors is that DNA molecular described in claim 2 or 3 is replaced to the LEA fragment in expression vector Bin19-35S-LEA-polyA, obtains expressing the recombinant vectors of albumen described in claim 1.
6. the primer pair of DNA molecular total length or its any fragment described in amplification claim 2 or 3.
7. recombinant vectors, expression cassette, transgenic cell line or the application of recombinant bacterium in regulating plant resistance of reverse described in DNA molecular or claim 4 described in albumen, claim 2 or 3 described in claim 1;
Described resistance of reverse is specially drought tolerance;
Described plant is specially dicotyledons or monocotyledons.
8. cultivate a method for transgenic plant, for the DNA molecular of albumen described in coding claim 1 is imported to object plant, obtain transgenic plant, the resistance of reverse of described transgenic plant is higher than described object plant.
9. method according to claim 8, is characterized in that: described resistance of reverse is drought tolerance;
Described in described coding claim 1, the DNA molecular of albumen imports object plant by the recombinant vectors described in claim 4 or 5.
10. method according to claim 8 or claim 9, is characterized in that:
Described object plant is dicotyledons or monocotyledons.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105669850A (en) * | 2016-04-15 | 2016-06-15 | 中国科学院遗传与发育生物学研究所 | AtVDAC3 protein and application of coding genes of AtVDAC3 protein in cultivation of stress resistance plants |
| CN106011145A (en) * | 2015-09-14 | 2016-10-12 | 中国科学院烟台海岸带研究所 | Stress-inducible gene deriving from jerusalem artichoke, encoded protein and application thereof |
| CN108866054A (en) * | 2017-05-08 | 2018-11-23 | 南京农业大学 | A kind of cotton non-coding RNA GhDAN1 and its application |
| CN114702563A (en) * | 2020-12-16 | 2022-07-05 | 中国农业大学 | Application of protein GRMZM2G088112 in regulation and control of plant drought resistance |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101798341A (en) * | 2009-02-11 | 2010-08-11 | 中国科学院植物研究所 | Heat shock factor protein relevant to resistance of plants as well as coding gene and application thereof |
-
2013
- 2013-04-26 CN CN201310149496.4A patent/CN104119430B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101798341A (en) * | 2009-02-11 | 2010-08-11 | 中国科学院植物研究所 | Heat shock factor protein relevant to resistance of plants as well as coding gene and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| GUOQIANG JIANG等: "Proteome analysis of leaves from the resurrection plant boea hygrometrica in respones to dehydration and rehydration", 《PLANTA》 * |
| SHAO HONG-BO等: "Lea protein in higher plants:structure, function, gene expression and regulation", 《COLLOIDS AND SURFACES B:BIOINTERFACES》 * |
| YAN ZHU等: "Ectopic over-expression of BhHsf1, a heat shock factor from the resurrection plant Boea hygrometrica, leads to increased thermotolerance and retarded growth in transgenic arabidopsis and tobacco", 《PLANT MOL BIOL.》 * |
| 刘霞 等: "复苏植物牛耳草BhLEA2基因启动子的分离和基因表达模式研究", 《自然科学进展》 * |
| 无: "Seed maturation protein PM41[Glycine max]", 《GENBANK》 * |
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|---|---|---|---|---|
| CN106011145A (en) * | 2015-09-14 | 2016-10-12 | 中国科学院烟台海岸带研究所 | Stress-inducible gene deriving from jerusalem artichoke, encoded protein and application thereof |
| CN106011145B (en) * | 2015-09-14 | 2019-07-05 | 中国科学院烟台海岸带研究所 | A kind of adversity gene and its coding albumen and application from jerusalem artichoke |
| CN105669850A (en) * | 2016-04-15 | 2016-06-15 | 中国科学院遗传与发育生物学研究所 | AtVDAC3 protein and application of coding genes of AtVDAC3 protein in cultivation of stress resistance plants |
| CN108866054A (en) * | 2017-05-08 | 2018-11-23 | 南京农业大学 | A kind of cotton non-coding RNA GhDAN1 and its application |
| CN108866054B (en) * | 2017-05-08 | 2021-08-10 | 南京农业大学 | Cotton non-coding RNA gene GhDAN1 and application thereof |
| CN114702563A (en) * | 2020-12-16 | 2022-07-05 | 中国农业大学 | Application of protein GRMZM2G088112 in regulation and control of plant drought resistance |
| CN114702563B (en) * | 2020-12-16 | 2023-03-24 | 中国农业大学 | Application of protein GRMZM2G088112 in regulation and control of plant drought resistance |
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